Roegneria yenchiana: A new species in the Triticeae (Poaceae) from the Hengduan Mountain region

Abstract Roegneria yenchiana sp. nov. (Triticeae) is a new species collected from Shangri‐la of Yunnan Province in China based on morphological, cytological, and molecular data. It is morphologically characterized by one spikelet per node, rectangular glums, awns flanked by two short mucros in lemmas, distinguished from other species of Roegneria. The genomic in situ hybridization results indicate that R. yenchiana is an allotetraploid, and its genomic constitution is StY. Phylogenetic analyses based on multiple loci suggested that R. yenchiana is closely related to Pseudoroegneria and Roegneria, and the Pseudoroegneria served as the maternal donors during its polyploid speciation.

tribe (Poaceae: Triticeae), which includes approximately 100 species that are of Asian origin and distributed in the Qinghai-Tibetan Plateau, Central Asia, East Asia, West Asia and South-east Europe (Yen & Yang, 2011).The largest number is found mainly in the mountains of the Qinghai-Tibetan Plateau, with 33 species being recorded.Most species of Roegneria can usually be recognized in the field by a combination of characters: (a) lack of underground rhizomes; (b) spikes often slightly curved to erect, with arrangement of spikelets nearly parallel to and appressed to rachis and only one per node, with long rachis internodes; (c) spikelets functionally disarticulating above the glumes (Baum et al., 1995).

| Plant sampling
Three accessions of the new species were sampled, with each accession representing one sample point based on distinct locality and/or altitude (Table S1).Twelve StY genome species from the genus Roegneria and 33 diploid taxa representing 19 basic genomes in Triticeae were included in this study, and their DNA sequences for phylogenetic analyses were obtained from published data (Fan et al., 2013;Huang et al., 2002;Lei et al., 2022;Mason-Gamer, 2004) (Table S1).The plants and voucher specimens are deposited at the Herbarium of Triticeae Research Institute, Sichuan Agricultural University, China (SAUTI).

| Morphological observation
Morphological features including underground rhizomes, stalks, cauline internodes, leaf auricles and blades, glumes, lemmas, paleas, awns, and anthers were observed.At least 20 measurements on fresh material were performed on each morphological variable by using a stereomicroscope (Olympus SZX7, Tokyo, Japan) with a digital camera.

| Genomic in situ hybridization (GISH)
Roots of the new species were collected from germinating seeds and adult plants, treated with nitrous oxide for 2.5 h, and fixed in 90% glacial acetic acid for 5 min.Chromosome preparations were performed using drop methods, according to Tan et al. (2021).The genomic DNA was isolated from fresh leaves by the CTAB method and then labeled with the DIG-Nick Translation Kit (Roche, Indianapolis, IN, USA).Roegneria ciliaris with the StY genomic constitution was used as a probe.The hybridization procedure, detection, and visualization methods follow those described by Tan et al. (2021).Images were captured with an Olympus BX51 fluorescence microscope (Japan).

| Phylogenetic analysis
Phylogenetic analysis is routinely applied to illustrate evolutionary and taxonomic questions.We carried out phylogenetic analysis for the new species and its affinitive species within the Triticeae based on three unlinked single-copy nuclear genes (Acc1, plastid Acetyl-CoA carboxylase; DMC1, disrupted meiotic cDNA; GBSSI, Granule-Bound Starch Synthase I) and three chloroplast regions [trnL-F, trnL (UAA)-trnF (GAA); matK, maturase coding gene; rbcL, ribulose-1, 5-bisphosphate carboxylase/oxygenase].Prior to phylogenetic analysis, The Acc1, DMC1, GBSSI, trnL-F, matK, and rbcL sequences were amplified by polymerase chain reaction (PCR) using the primers listed in Table S2 under cycling conditions reported previously (Sha et al., 2016(Sha et al., , 2017)), and PCR products were cloned into the pMD18-T vector (TaKaRa, Dalian, China) following the manufacture's instruction.At least 10 random independent clones were selected for commercial sequencing.For each gene fragment, in cases when multiple identical sequences resulted from cloned PCR products of each accession, only one sequence was included in the data matrix.
Multiple sequence alignment was conducted using ClustalX (Thompson et al., 1999), with default parameters and additional manual edits to minimize gaps.Phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian inference (BI).ML analysis was performed using RAxML v8.2.8 under the GTR + GAMMA model on the XSEDE supercomputer at the CIPRES Science Gateway platform (Miller et al., 2010).Analyses included inference of the 'best tree' and generation of 1000 bootstrap replicates to obtain node support measures.BI analysis was conducted with MrBayes v3.2.7a under the same evolutionary model and supercomputer platform (Miller et al., 2010) as ML analysis.Four MCMC (Markov Chain Monte Carlo) chains were run for 2,000,000 generations.Trees were sampled every 1000 generations until reaching the convergence parameters (standard deviation less than 0.01).The first 25% of generated trees representing the burn-in phase were discarded, and the remaining trees were used to construct the 50%-majority rule consensus trees.
The statistical confidence in nodes was evaluated by posterior probabilities (PP).PP-value less than 90% was not included in the figures.

| Morphological characters
Roegneria yenchiana is morphologically recognized as a species of Roegneria by the typical characters that have traditionally one spikelet per node and spikelets with functionally disarticulating above the glumes.This species is distinguished from other species of Roegneria by its rudimentary spikelets at the tortuous inflorescence base, rectangular glumes, and awns flanked by two short mucros in lemmas (Figure 1a-d).Roegneria ciliaris occurring in the Hengduan Mountain region has the StY genomes (Lu, 1992), which is morphologically the most similar species to R. yenchiana.Despite this, R. ciliaris has not been observed near to the place where R. yenchiana grows.
Roegneria yenchiana can be distinguished from R. ciliaris in several traits, including glume length, ciliates in lemma margin, lemma back, and the paleas length.

| GISH
GISH was carried out with the StY genome species (from Roegneria ciliaris) being used as a probe.GISH results showed that R. yenchiana has 28 chromosomes, and all the chromosomes displayed StY genome hybridization signal along their entire chromosomal length (Figure 1e).Thus, the genomic constitution of R. yenchiana is StStYY.

| Phylogenetic analyses
Three unlinked low-copy nuclear gene (Acc1, DMC1, and GBSSI) and three chloroplast region (trnL-F, matK, and rbcL) sequences were separately amplified and sequenced for all the accessions of R. were variable characters and 132 were informative.ML and Bayesian analyses of the combined chloroplast data recovered the same topology.In the ML tree (−Lnlikelihood = 7088.8370)inferred from the combined chloroplast data, all the accessions of R. yenchiana formed one monophyletic group, and this group was clustered with the species from Roegneria and Pseudoroegneria (Figure S3).

| Diagnosis
Roegneria yenchiana is morphologically the most similar species to R. ciliaris (StY) but is distinguished from R. ciliaris by its lower glume length (3-5 mm vs. 7-8 mm), ciliates in lemma margin (absent vs. about 1 mm), lemma back (scabrous or pubescent on lower parts vs. hispid), and the paleas length (slightly shorter than lemma vs. 2/3 the length of lemma).

| DISCUSS ION
The morphological characters of R. yenchiana differ from all species previously described in Roegneria, especially in its rudimentary spikelets at the inflorescence base, rectangular glumes, and two short mucros in lemmas.GISH results confirmed that R. yenchiana is a tetraploid (2n = 4x = 28) with the StY genome constitution.Based on the genomic system of classification as a guide to resolve taxonomic treatment in Triticeae (Barkworth & von Bothmer, 2009;Baum et al., 1995;Dewey, 1984;Wang et al., 1994;Yen & Yang, 2011), this species should be included in the genus Roegneria.Phylogenetic analysis based on all three unlinked single-copy sequences showed that the Stgenome homoeologous types of R. yenchiana were grouped with the sequences of Pseudoroegneria and Roegneria, and the Ygenome homoeologous types of R. yenchiana were clustered with the sequences of Roegneria with high statistic supports (BS > 70% and PP > 0.9).In the phylogenetic tree inferred from the combined chloroplast sequences, all three accessions of R. yenchiana were grouped with Pseudoroegneria and Roegneria (BS = 81% and PP = 1.0).These considered that the Y genome has its origin in Central Asia or the Himalaya region, and may be extinct.Extensive cytogenetic and molecular studies have suggested that the St (Pseudoroegneria), W [Australopyrum (Tzvelev) Á. Löve], V [Dasypyrum villosum (L.) Candargy], and Xp [Peridictyon O. Seberg, S. Frederiksen & C. Baden] genomes are potential donors of the Y genome Here, we described a new species of Roegneria discovered from the Hengduan Mountain Region during an expedition to the Qinghai-Tibetan plateau in 2021 to collect germplasm of various members of the Triticeae.The new species was discovered in stony slope of Haba Snow Mountain at altitude about 3200 m and 2500 m, and in stony slope of Yulong Snow Mountain at an altitude of approximately 3200 m.The two localities are more than 90 km apart.Using multiple sampling of the new species, we recognized it with morphological observations, genomic in situ hybridization (GISH), and phylogenetic reconstruction methods.Our goal was to determine whether this was the case as a new species within Roegneria and, if so, to determine the genomic constitution of the new species and provide it with an appropriate name.
yenchiana.They were then analyzed with published Stand Ytype sequences from twelve species of Roegneria and those from 33 diploid taxa representing 19 basic genomes in Triticeae.Consequently, six homoeologous sequences representing two distinct types (Stand Ytype) of each nuclear gene and three sequences of each chloroplast region were detected from all the accession of R. yenchiana.Four datasets, including Acc1 data, DMC1 data, GBSSI data, and combined chloroplast (trnL-F + matK + rbcL) data, were used to conduct separately alignments and phylogenetic analyses.The features of Acc1 data, DMC1 data, GBSSI data, and combined chloroplast data were summarized in Table S3.The aligned Acc1 sequences yielded 1400 characters, of which 411 were variable characters and 201 were informative.ML analysis of the Acc1 data yielded a single phylogenetic tree (−Lnlikelihood = 5890.5424).ML and Bayesian analyses of the Acc1 data recovered the same topology.The tree illustrated in Figure 2 was the ML tree of bootstrap support (BS) above and posterior probabilities (PP) below branches.The Acc1 sequences of R. yenchiana were represented in two clades, corresponding to the two genomic types (St and Y).The Sttype Acc1 sequences of R. yenchiana were in one monophyletic group and then formed a paraphyletic grade with those Sttype sequences from F I G U R E 1 Features of Roegneria yenchiana.(a) Inflorescences; (b) Glumes; (c) Lemmas; (d) Palea; (e) Genomic in situ hybridization photographs of mitotic metaphase in Roegneria yenchiana display red fluorescence when probed with R. ciliaris, indicating R. yenchiana has the StY genomes.F I G U R E 2 Phylogenetic tree inferred from the Acc1 sequences of Roegneria yenchiana and its affinitive species within the Triticeae.Numbers above nodes are bootstrap support values ≥50%, and numbers below nodes are Bayesian posterior probability values ≥90%.Polyploid species names are followed by GenBank numbers.The upper case letters in parentheses indicate the genome type of the species.| 5 of 8 SHA et al. other Roegneria and Pseudoroegneria.The Ytype Acc1 sequences of R. yenchiana and the sequence of Roegneria ciliaris formed a paraphyletic grade.Of the 959 total characters in the DMC1 data, 343 characters were variable, and 158 characters were informative.ML and Bayesian analyses of the DMC1 data recovered the same topology.The ML tree (−Lnlikelihood = 4907.2580)inferred from the DMC1 data showed that the Sttype and Ytype sequences from the new species were split into two well-supported clades (Figure S1).The Sttype DMC1 sequences of R. yenchiana were scattered in different groups and clustered with those from different species of Roegneria.The Ytype DMC1 sequences of R. yenchiana formed one monophyletic group and grouped with the sequence of Roegneria brevipes.In the GBSSI sequence data matrix, of the 1104 total characters, 472 were variable, and 277 were parsimony informative.ML and Bayesian analyses of the GBSSI data recovered the same topology.In ML tree (−Lnlikelihood = 8424.8144)inferred from the GBSSI data, the Sttype GBSSI sequences of R. yenchiana formed one monophyletic group, and then clustered with one group including Roegneria gmelinii and Roegneria pendulinus (Figure S2).The Ytype GBSSI sequences of R. yenchiana were in one monophyletic group and formed a paraphyletic grade with those Ytype sequences from several species of Roegneria (R. semicostatus, R. anthosachnoides, and R. ciliaris).The combined trnL-F, matK, and rbcL data yielded 3089 characters of which 306 18-25 mm (excluding awns) long, with 6-10 florets, disarticulation above the glumes, beneath each floret.Glums unequal, rectangular, oblique, margin membranous, usually lower glume 2/3 the length of upper glume, lower glum 3-5 mm, upper glume 5-8 mm, 3-5 prominent veins, sometimes setulose on upper veins, apex acuminate and with 1 tooth.Lemmas oblong-lanceolate to oblong, scabrous, pubescent on lower parts, 3 veins, prominent midvein, first lemma 9-12 mm, awned, awns 17-25 mm, reflexed, awns flanked by 2 short teeth with about 0.5 mm long.Paleas oblong, slightly shorter than lemma, ciliolate along keels in the distal 1/3, apex truncate.Chromosome number 2n = 4x = 28; genome constitution StStYY.
3.4.4| Distribution, habitat, and nameRoegneria yenchiana is known only from a few localities in the northwest of Yunnan, China.It was found among bushes or on stony mountain slopes between 2500 and 3500 m, growing together with Campeiostachys nutans (Griseb.)J. L. Yang, B. R. Baum et C. Yen and Campeiostachys dahurica var.cylindrica (Franch.)J. L. Yang, B. R. Baum et C. Yen.These two species were previously included in the genus Elymus.According to the genome system of classification, they were transferred into the genus Campeiostachys because of their StYH genome constitutions.Roegneria yenchiana is named for commemorating Prof. Yen Chi, a biosystematic scientist of the Triticeae from the Triticeae Research Institute of Sichuan Agricultural University (SAU), China.He was evaluated as an important person in the taxonomic research of the Triticeae by Chairman Prof. Roland von Bothmer in the eighth international Triticeae symposium.
results indicated that Pseudoroegneria and Roegneria are closely related to R. yenchiana.Combined with GISH analysis, it can be concluded that the St genome of R. yenchiana is derived from the genus Pseudoroegneria, and the Pseudoroegneria served as the maternal donors during its polyploid speciation.AUTH O R CO NTR I B UTI O N S Li-Na Sha: Conceptualization (equal); writing -original draft (equal); writing -review and editing (equal).Xiao Liang: Investigation (lead); methodology (equal).Xin-Yi Zhang: Investigation (equal); methodology (equal).Shan Gao: Investigation (equal); methodology (equal).Yue Zhang: Investigation (equal); methodology (equal).Yong-Hong Zhou: Conceptualization (equal); writing -review and editing (equal).

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The holotype of the new species Roegneria yenchiana X. Fan et L. N. Sha.